Adsorption-loaded silk nanoparticles exploit protein electrostatics and β-sheet-rich architecture to achieve high doxorubicin loading and pH-responsive release, while preserving silk structure. Ensemble and single-particle analyses reveal exceptional formulation uniformity, while in vitro studies confirm preserved cytotoxicity against MDA-MB-231 human breast cancer cells.

The brain has limited spontaneous tissue regeneration capacity after stroke, partly due to the absence of an extracellular matrix in the stroke microenvironment. Self-assembling silk fibroin hydrogels can serve as a tissue-mimetic extracellular matrix; however, more information is needed on their behavior in the chronic stroke setting. We hypothesized that in the chronic stroke setting, self-assembling silk fibroin hydrogels serve as a reliable support matrix for regeneration in the stroke cavity. In this study, male Sprague-Dawley rats (240-290 g, 8-9 weeks old (n = 8) underwent transient middle cerebral artery occlusion 2 weeks before stereotactic injection of 4% w/v self-assembling silk fibroin hydrogels into the stroke cavity. Animals were randomly assigned to be terminated at 6- and 12-months postimplantation (n = 4/group) for blinded immunohistological analysis of the in situ distribution of the silk hydrogels and cellular infiltration and characterization. Results showed that robust in situ gelation with a good hydrogel-host tissue interface was observed with hydrogel remnants still evident at 1-year postgrafting. At 6 months postgrafting, most cellsprimarily astrocytes and microglia/macrophageswere localized at the tissue-hydrogel interface and were CD206+ expressing, whereas the cells that substantially infiltrated the center of the hydrogels at 12 months showed a hybrid of CD86+ and CD206+ phenotypes. The hydrogel areas surrounded by macrophages showed evidence of degradation, potentially providing a niche for endogenous neuronal progenitor cell proliferation and migration (DCX+/Ki67+) that was evident in the hydrogels. These findings showed that self-assembling silk fibroin hydrogels effectively induce phenotypic changes in microglia and macrophages chronically after stroke that might favor tissue neurogenesis. These are important features for the development of next-generation stroke therapies.

Silk hydrogels have attracted increasing interest as biocompatible substrates for neural cell culture and regenerative research. Recent research has shown that activation of exchange protein directly activated by cAMP 2 (Epac2) promotes neurite outgrowth in several neuronal models. Here, we investigated the effects of Bombyx mori silk hydrogel, alone or in combination with S-220, a non-hydrolysable cAMP analogue that selectively activates Epac2, on neurite outgrowth. Epac2 activation significantly and dose-dependently enhanced both the total and longest neurite length. Neurons cultured on silk hydrogel alone exhibited neurite outgrowth comparable to control neurons, indicating good biocompatibility of the hydrogel. Addition of S-220 to the culture medium with silk hydrogel modestly improved neurite morphology; however, incorporation of S-220 directly into the hydrogel did not enhance neurite outgrowth but instead reduced neurite length. A subsequently established three-dimensional cortical neuron culture model using silk hydrogel supported neurite extension and increased branching complexity beyond that observed with two-dimensional cultures, despite a shorter overall neurite length. Collectively, these findings support silk hydrogel as a permissive biomaterial substrate for in vitro cortical neuron culture. The system provides a useful platform for investigating neurite outgrowth, neuron-biomaterial interactions and the delivery of bioactive compounds in two- and three-dimensional neural cultures.

The increasing application of nanomedicine requires deeper understanding of the interaction of the carrier with the circulatory system, as this system often serves as the gateway for carrier distribution in the body. As foreign materials, nanoparticles can activate blood coagulation and inflammatory systems. However, whether these activation processes respond linearly to the particle count, the interface area with blood, or the total mass remains uncertain. This study incubated 115, 240 or 450 nm diameter silk nanoparticles in vitro in flowing whole human blood at concentrations matching the total mass, total surface area of ideal spherical particles, and particle count and then analyzed the activation of the coagulation cascade, blood platelets, complement cascade and granulocytes. The immunocell association of nanoparticles was highly modulated by plasma proteins, which had a passivating effect. The activation of the humoral cascades mainly depended on the applied mass concentration, whereas granulocyte activation tended to show linear dependence mainly on the particle count, suggesting a direct interaction effect. Additional factors, such as curvature and area restriction for the assembly of enzyme complexes, superposed these effects on basic geometric parameters. Thus, the design of silk nanoparticles for drug delivery has to prioritize either low cell activation or low activation of plasmatic pathways.

Silk has emerged as a promising biomaterial for formulating protein-based nanocarriers for use as drug delivery systems. Silk nanoparticles can be manufactured using several different methods, including nanoprecipitation through organic desolvation using isopropanol as an antisolvent. The translation of silk nanoparticle manufacture from the bench to the industrial scale requires deeper insights into the manufacturing process, especially nanoparticle purification. Here, we compare the impact of tangential flow filtration (TFF) with widely used centrifugation methods for silk nanoparticle purification. Silk nanoparticles were manufactured using a well-studied semi-batch nanoprecipitation process. We demonstrate that silk nanoparticle purification significantly changed the physicochemical properties, particularly the yield. The silk nanoparticles showed major differences in in vitro cytotoxicity, depending on the purification method used. TFF purification revealed that a change in permeate volume during purification was a key parameter, as low cytotoxicity was correlated with an increase in the diavolume. Overall, TFF emerges as a purification method with high potential for scale-up and faster silk nanoparticle purification.

Silk fibroin is a promising biomaterial for nanocarrier-based drug delivery due to its biocompatibility, biodegradability, and tunable mechanical properties. In addition, the silk protein is amenable to various processing strategies, offering flexibility for optimizing particle characteristics. Emerging evidence highlights that metal ions can modulate silk conformation and structure in the silk gland, as well as influencing self-assembly, potentially impacting silk nanoparticle fabrication. Our previous study highlighted the potential of Ca2+ in silk nanoparticle fabrication. However, other metal ions in the silk gland influence silk fibroin behavior too. Here, we investigate how potassium ions (K+), with similar abundance to Ca2+ in the silkworm gland, influence silk nanoparticle formation as modulators of self-assembly and material properties, aiming to produce nanoparticles with distinct physicochemical profiles. We show that K+ enhances silk assembly, increases nanoparticle size, alters surface charge (zeta potential), and boosts production yield, thereby minimizing silk wastage during silk nanoparticle preparation. Potassium ions also significantly improve payload encapsulation efficiency, making K+ inclusion valuable for a range of drug-loading applications. The resulting silk nanoparticles exhibit reduced toxicity and inflammatory response, highlighting their promise as safe and effective nanocarrier candidates for drug delivery. Our findings establish K+ as a fundamental yet powerful tool for tuning silk nanoparticle properties to meet pharmaceutical needs.

Silk fibroin is a promising material for nanocarrier-based drug delivery applications due to its biocompatibility, biodegradability, and mechanical properties, which can be fine-tuned through processing conditions. In this study, we explore the impact of Ca2+ and K+ inclusion on the morphology of silk nanoparticles and evaluate the short- and long-term stability of silk nanoparticles formed by antisolvent precipitation in deionized water and sodium phosphate buffer. Using advanced electric asymmetric flow field-flow fractionation multiplexed with online detectors (EAF4-UV-MALS-DLS) and orthogonal analytics (DLS, ELS, NTA, FE-SEM), we analyze the physicochemical attributes of silk nanoparticles. We find significant differences in nanoparticle architecture and stability in different buffers, with notable differences in particle size (R g and R h), charge, and shape measured over 56 days. Notably, nanoparticles formulated with 0.7 mg Ca2+ and 1.1 mg K+ maintained superior physicochemical stability, making them promising candidates for future nanocarrier-based applications.

Silk has emerged as an interesting candidate among protein-based nanocarriers due to its favorable properties, including biocompatibility and a broad spectrum of processing options to tune particle critical quality attributes. The silk protein conformation during storage in the middle silk gland of the silkworm is modulated by various factors, including the most abundant metallic ion, calcium ion (Ca2+). Here, we report spiking of liquid silk with calcium ions to modulate the silk nanoparticle size. Conformational and structural analyses of silk demonstrated Ca2+-induced silk assemblies that resulted in a liquid crystalline-like state, with the subsequent generation of β-sheet-enriched silk nanoparticles. Thioflavin T studies demonstrated that Ca2+ effectively induces self-assembly and conformation changes that also increased model drug loading. Ca2+ incorporation in the biopolymer feed significantly increased the nanoparticle production yield from 16 to 89%, while simultaneously enabling Ca2+ concentration-dependent particle-size tuning with a narrow polydispersity index and altered zeta potential. The resulting silk nanoparticles displayed high biocompatibility in macrophages with baseline levels of cytotoxicity and cellular inflammation. Our strategy for manufacturing biomimetic silk nanoparticles enabled overall tuning of particle size and improved yields─features that are critical for particle-based nanomedicines.

This review introduces nanomedicines and medical silks by addressing seminal and recent research within these fields. First, the successes of nanoparticles in improving the safety profiles and pharmacokinetic–pharmacodynamic properties are explored but also the concepts of threshold dosing and targeting of tumor-associated macrophages. Current barriers to systemic delivery of nanomedicines are detailed and methods to overcome these barriers and increase tumor targeting are evaluated, namely: tuning the nanomedicine size and surface charge for enhanced tumor accumulation and penetration; non-spherical nanomedicine morphologies for macrophage evasion and targeted delivery to endothelial cells; and, surface functionalization for stealth coatings and targeting receptor-mediated endocytosis. The advantages of using silk as a nanomedicine with reference to its structure, composition, biological performance, and formulation are discussed. While batch methods for silk processing enable the formation of nano to microparticles, continuous technology can overcome bottlenecks of the deployed engineering methods such as low throughput and poor reproducibility. Finally, the chemical modification of silk using homogeneous and heterogenous chemistries is assessed within the nanomedicine context. Overall, this review covers silk nanomedicines from first principles to carrier design and on to areas of future development.

Biopolymer silk has a long tradition of use in human health. However, our ability to unspin the silk fiber, in combination with our emerging understanding of the silk self-assembly process, now enables us to exploit silk for new applications, including silk nanomedicines. While the nanomedicine field is rapidly coming of age, a need remains to learn from past lessons to inform current and future silk nanomedicine research. This chapter provides a brief background on the use of polymers in nanomedicines. This information subsequently sets the stage for a selected but critical examination of silk processing, silk nanoparticle manufacture, and assessment.

To overcome the precision limitation and environmental impact of current chemical-based production methods for manufacturing silk microfibres used for targeted drug delivery, this paper presents a high-precision, scalable, eco-friendly mechanical machining approach to produce such microfibres in the form of discontinuous chips obtained through elliptical vibration turning of silk fibroin film using a diamond tool. The length and waist width of fabricated microfibres can be precisely controlled. As each vibration cycle will produce one silk microfibre, complete and deterministic chip breakage becomes an essential and challenging task in this approach due to its unique two-phase structure. Thus, the hybrid FE-SPH numerical simulations and machining experiments were conducted to gain a pioneering and in-depth exploration of the chip-breaking mechanism in this process. It was found that applying a low depth ratio (ratio of the nominal depth of cut to the tool path vertical amplitude) and a high horizontal speed ratio (the nominal cutting speed versus the critical workpiece velocity) could effectively reduce the average tool velocity angle (the angle from the deepest cut to the tool exit point along the cutting direction). A smaller angle would enhance the diamond tool's shearing action and led to the reduction of hydrostatic pressure in the cutting zone and a consequent decrease in the ductility of silk fibroin due to its unique structure dominated by beta-sheet crystallites. The above adjustments collectively facilitated chip breakage. This paper, therefore, established a governing rule for the controlled and repeatable formation of microfibres based on the average tool velocity angle for the first time and revealed that the cutting chips would undergo complete and deterministic breakages once the angle approached below 22.6°. On this basis, the high-precision and scalable manufacturing of silk microfibres with precisely controllable length and waist width was ultimately achieved.

Antibacterial properties are desirable in wound dressings. Silks, among many material formats, have been investigated for use in wound care. However, the antibacterial properties of liquid silk are poorly understood. The aim of this study is to investigate the inherent antibacterial properties of a Bombyx mori silk fibroin solution. Silk fibroin solutions containing ≥ 4% w/v silk fibroin do not support the growth of two common wound pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. When liquid silk is added to a wound pad and placed on inoculated culture plates mimicking wound fluid, silk is bacteriostatic. Viability tests of the bacterial cells in the presence of liquid silk show that cells remain intact within the silk but could not be cultured. Liquid silk appears to provide a hostile environment for S. aureus and P. aeruginosa and inhibits growth without disrupting the cell membrane. This effect can be beneficial for wound healing and supports future healthcare applications for silk. This observation also indicates that liquid silk stored prior to processing is unlikely to experience microbial spoilage.

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Despite many reports detailing silk hydrogels, the development of composite silk hydrogels with homotypic and heterotypic silk nanoparticles and their impact on material mechanics and biology have remained largely unexplored.

Silk hydrogels have shown potential for tissue engineering applications, but several gaps and challenges, such as a restricted ability to form hydrogels with tuned mechanics and structural features, still limit their utilisation. Here, Bombyx mori and Antheraea mylitta (Tasar) silk microfibres were embedded within self-assembling B. mori silk hydrogels to modify the bulk hydrogel mechanical properties. This approach is particularly attractive because it creates structured silk hydrogels. First, B. mori and Tasar microfibres were prepared with lengths between 250 and 500 μm. Secondary structure analyses showed high beta-sheet contents of 61% and 63% for B. mori and Tasar microfibres, respectively. Mixing either microfibre type, at either 2% or 10% (w/v) concentrations, into 3% (w/v) silk solutions during the solution–gel transition increased the initial stiffness of the resulting silk hydrogels, with the 10% (w/v) addition giving a greater increase. Microfibre addition also altered hydrogel stress relaxation, with the fastest stress relaxation observed with a rank order of 2% (w/v) > 10% (w/v) > unmodified hydrogels for either fibre type, although B. mori fibres showed a greater effect. The resulting data sets are interesting because they suggest that the presence of microfibres provided potential ‘flow points’ within these hydrogels. Assessment of the biological responses by monitoring cell attachment onto these two-dimensional hydrogel substrates revealed greater numbers of human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) attached to the hydrogels containing 10% (w/v) B. mori microfibres as well as 2% (w/v) and 10% (w/v) Tasar microfibres at 24 h after seeding. Cytoskeleton staining revealed a more elongated and stretched morphology for the cells growing on hydrogels containing Tasar microfibres. Overall, these findings illustrate that hydrogel stiffness, stress relaxation and the iPSC-MSC responses towards silk hydrogels can be tuned using microfibres.

Medical silks have captured global interest. While silk sutures have a long track record in humans, silk bioconjugates are still in preclinical development. This perspective examines key advances in silk bioconjugation, including the fabrication of silk-protein conjugates, bioconjugated silk particles, and bioconjugated substrates to enhance cell-material interactions in two and three dimensions. Many of these systems rely on chemical modification of the silk biopolymer, often using carbodiimide and reactive ester chemistries. However, recent progress in enzyme-mediated and click chemistries has expanded the molecular toolbox to enable biorthogonal, site-specific conjugation in a single step when combined with recombinant silk fibroin tagged with noncanonical amino acids. This perspective outlines key strategies available for chemical modification, compares the resulting silk conjugates to clinical benchmarks, and outlines open questions and areas that require more work. Overall, this assessment highlights a domain of new sunrise capabilities and development opportunities for silk bioconjugates that may ultimately offer new ways of delivering improved healthcare.

Silk fibroin nanoprecipitation by organic desolvation in semi-batch and microfluidic formats provides promising bottom-up routes for manufacturing narrow polydispersity, spherical silk nanoparticles. The translation of silk nanoparticle production to pilot, clinical, and industrial scales can be aided through insight into the property drifts incited by nanoprecipitation scale-up and the identification of critical process parameters to maintain throughout scaling. Here, we report the reproducibility of silk nanoprecipitation on volumetric scale-up in low-shear, semi-batch systems and estimate the reproducibility of chip parallelization for volumetric scale-up in a high shear, staggered herringbone micromixer. We showed that silk precursor feeds processed in an unstirred semi-batch system (mixing time > 120 s) displayed significant changes in the nanoparticle physicochemical and crystalline properties following a 12-fold increase in volumetric scale between 1.8 and 21.9 mL while the physicochemical properties stayed constant following a further 6-fold increase in scale to 138 mL. The nanoparticle physicochemical properties showed greater reproducibility after a 6-fold volumetric scale-up when using lower mixing times of greater similarity (8.4 s and 29.4 s) with active stirring at 400 rpm, indicating that the bulk mixing time and average shear rate should be maintained during volumetric scale-up. Conversely, microfluidic manufacture showed high between-batch repeatability and between-chip reproducibility across four participants and microfluidic chips, thereby strengthening chip parallelization as a production strategy for silk nanoparticles at pilot, clinical, and industrial scales.

Stroke is an unmet clinical need with a paucity of treatments, at least in part because chronic stroke pathologies are prohibitive to 'first-generation' stem cell-based therapies. Hydrogels can remodel the hostile stroke microenvironment to aid endogenous and exogenous regenerative repair processes. However, no clinical trials have yet been successfully commissioned for these 'second-generation' hydrogel-based therapies for chronic ischaemic stroke regeneration. This review recommends a path forward to improve hydrogel technology for future clinical translation for stroke. Specifically, we suggest that a better understanding of human host stroke tissue-hydrogel interactions in addition to the effects of scaling up hydrogel volume to human-sized cavities would help guide translation of these second-generation regenerative stroke therapies.

Origami folding is an easy, cost-effective, and scalable fabrication method for changing a flat material into a complex 3D functional shape. Here, we created semicrystalline silk films doped with iron oxide particles by mold casting and annealing. The flat silk films could be loaded with natural dyes and folded into 3D geometries using origami principles following plasticization. They performed locomotion under a magnetic field, were reusable, and displayed colorimetric stability. The critical parameters for the design of the semi-autonomous silk film, including ease of folding, shape preservation, and locomotion in the presence of a magnetic field, were characterized, and pH detection was achieved by eye and by digital image colorimetry with a response time below 1 min. We demonstrate a practical application-a battery-free origami silk boat-as a colorimetric sensor for waterborne pollutants, which was reusable at least five times. This work introduces silk eco-sensors and merges responsive actuation and origami techniques.